Team:DTU-Denmark/Project/POC MAGE
Methods
All the strain were made by homologous recombineering. Plasmids containing cassettes that were able to do a double-crossover homologous recombineering into the genome of B. subtilis 168 (referred to as knockout (KO) plasmid). These were used to, simultaneously, delete the desired gene (amyE or mutS) and inserting the expression cassette for one of the recombinases: beta or GP35.
Four different plasmids was assembled to make the four MAGE ready strains:
pDG268neo_Beta-neoR
pDG268neo_GP35-neoR
pSB1C3_Beta-neoR
pSB1C3_GP35-neoR
Missing: figure tekst: Shows the general concepts of the two plasmids pDG268neo_recombinase and pSB1C3_recombinase. Both exists in two versions, one with each of the recombinase proteins CDSs (Beta and GP35). Those also have different RBSs since they are optimized for the CDS. Upstream of neoR is a promoter and RBS and downstream of neoR is a terminator, but sequences and positions of these features are not known.
For pDG268neo_recobinase a DNA sequence containing following features
Missing: make a table on following
● Promoter: PliaG from BBa_K823002 was used.
● RBSs were optimized for the specific CDS using the salis lab RBS calculator (Missing https://www.denovodna.com/software/)
● CDS for recombination protein beta or GP35
● Terminator: we use rho-independent Part:BBa_B0014
Sequence was ordered from IDT as two gblocks (for each recombinase) with overlapping regions, thus they can be assembled with Gibsom assembly.
pDG268neo was linearized in a PCR with primers, so that the native lacZ was omitted. This linearized plasmid was purified and used in a Gibsom assembly reaction with two cognate gblocks. This is shown on the figure missing.
Missing figure text: Gibsom assembly of the pDG268neo_GP35, the gblocks was fused to the “gp35 Gblocks fused” in the same reaction. The Gibsom assembly of pDG268neo_beta was similar.
This resulted in two different plasmids pDG268neo_beta and pDG268neo_gp35.
The two mutS KO plasmids was made of the following DNA fragments:
1. About 500 bp up- and downstream from mutS was amplified, using primers with tails.
2. Recombinase and neoR expression cassettes was amplified from the pDG268neo_recombinase
3. Linearized pSB1C3 was used as template.
These fragments was assembled into two different plasmids pSB1C3_mutS::beta-neoR and pSB1C3_mutS::gp35-neoR using Gibsom assembly.
Missing figure text: Gibsom assembly of the pSB1C3_beta. The feature called “mutL” corresponding mutS downstream. The Gibsom assembly of pSB1C3_GP35 was similar.
All plasmids were verified by restriction enzyme digestion.
We prepared naturel competent B. subtilis 168 to be transformed. The plasmids were linearized with restriction enzymes. Then transformed into naturally competent B. subtilis 168, transformants were selected on 5ɣ neo. Transformants were verified with colony PCRs.
Department of Systems Biology
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